A recombinant virus carrying a foreign DNA insert may be used to deliver genes to cells, where the gene may be expressed, if desired, to permit production of recombinant proteins in vitro or in vivo, vaccination of human and non-human mammals, or treatment or amelioration of diseases or genetic defects in humans or in non-human mammals. One may treat or ameliorate diseases or genetic defects by providing some effective level of normal gene products, increased levels of gene products or by blocking endogenous production of a gene, whose expression would be deleterious to the cell or organism.
Methods for delivering an exogenous gene to a mammalian cell include the use of mammalian viral vectors, such as those that are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, adeno-associated viruses, hybrid viruses (e.g., hybrid adenovirus-AAV, see U.S. Pat. No. 5,856,152) and the like. Other methods include direct injection of DNA, biolistic administration of DNA, electroporation, calcium phosphate precipitation, as well as methods of administration which utilize ligand-DNA conjugates, liposome conjugates of DNA, polycation-DNA complexes or adenovirus-ligand-DNA conjugates.
Adeno-associated virus (AAV) systems have many advantages that can be exploited for delivery of transgenes. AAV is a helper-dependent DNA parvovirus which belongs to the genus Dependovirus. AAV requires helper function in order for a productive infection to occur. Helper functions may be provided by a number of agents, but generally co-infection with an unrelated helper virus, either adenovirus, herpesvirus or vaccinia, is used. In the absence of such co-infection, AAV establishes a latent state by insertion of its genome into a host cell chromosome. Subsequent infection by a helper virus rescues the integrated copy which can then replicate to produce infectious viral progeny. AAV has a wide host range and is able to replicate in cells from any species so long as there is also a successful co-infection of such cells with a suitable helper virus. AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For a review of AAV, see, e.g., Berns and Bohenzky (1987) Advances in Virus Research (Academic Press, Inc.) 32:243–307.
AAV has a genome of about 4.7 kb in length, including inverted terminal repeats (ITRs) that often, but not necessarily are 145 nucleotides in length. The AAV genome encodes two genes, rep and cap, each of which expresses a family of related proteins from separate open reading frames and which are produced by alternative mRNA splicing and different transcriptional and translational start sites. Rep polypeptides (Rep78, Rep68, Rep52, and Rep40) are involved in replication, rescue and integration of the AAV genome. Rep78 and Rep68 have the same amino-terminal sequence and share the same promoter, p5, but Rep78 contains an exon that is alternatively spliced out in rep68. Similarly, Rep52 and Rep40 have the same amino-terminal sequence and share the p19 promoter, which is downstream from the p5 promoter, but rep52 contains an exon that is alternatively spliced out in rep68. Cap proteins (VP1, VP2, and VP3) form the virion capsid. Cap gene transcription is driven by the p40 promoter. See FIG. 2B for a schematic diagram of the rep and cap genes and promoters p5, p19 and p40. Flanking the rep and cap open reading frames at the 5′ and 3′ ends of the AAV genome are the ITRs. In certain AAV genomes, the ITRs are 145 nucleotides in length, the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. The entire nucleic acid encoding rep and cap can be excised and replaced with a transgene [B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155–168 (1990)]. The ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome if other sources of rep and cap are provided.
When AAV infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Upon introduction of helper functions into the cell, such as by infection with a helper virus, the AAV provirus is rescued and amplified. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or − single stranded DNA genomes following cell lysis.
Replacing the rep and cap sequences with a desired transgene yields a rAAV capable of delivering the transgene to target host cells. In current methods, the deleted rep and cap sequences are supplied to the host cells by other viruses or plasmids where they are transiently or stably expressed. There are also a number of cell lines that stably express rep and cap. The host cells also require helper functions in order for the rAAV to replicate and excise from the host cell genome. The helper functions usually are provided by helper viruses (either wildtype or crippled viruses), plasmids containing the helper virus functions or physical methods.
Although it is known that rep is required for replication and excision of AAV, the amount of Rep proteins required for effective rAAV production is, as yet, unclear. U.S. Pat. No. 5,354,678 states that Rep proteins may be toxic to certain cell lines, and WO 97/06272, WO 98/46728 and Li et al. suggest that attenuation of Rep78/68 production results in higher levels of production of rAAV. In contrast, other art, such as U.S. Pat. No. 5,658,776, explicitly states that high expression of Rep proteins—a result of replacing the native p5 promoter with a strong promoter, such as the human immunodeficiency virus long terminal repeat (HIV LTR)—results in high level expression of rAAV. Similarly, U.S. Pat. No. 5,837,484 states that the p5 promoter should be replaced by a strong constitutive promoter or inducible promoter, such as the metallothionein promoter, in order to overcome the strong feedback inhibition by Rep of its own transcription. Thus, U.S. Pat. Nos. 5,658,776 and 5,837,484 suggest that high expression of Rep78/68 is required for efficient rAAV production.
One method that has been used to produce recombinant AAV (rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid containing rAAV sequences (the cis plasmid) and a plasmid containing rep and cap (the trans plasmid), and infecting the cells with a helper virus (e.g., adenovirus or herpes virus). See U.S. Pat. No. 5,753,500. Li et al. (J. Virol. 71:5236–5243, 1997) have modified this method by altering the translation initiation codon of the Rep78/68 proteins in the trans plasmid to decrease the translation of the Rep protein and increase production of rAAV. However, the disadvantage of the methods taught by U.S. Pat. No. 5,753,500 and Li et al. is that co-transfection of two plasmids along with infection by a helper virus is inefficient, may exhibit poor reproducibility, may result in generation of pseudo-wildtype replication-competent AAV (rcAAV), and cannot be easily scaled up for industrial production of rAAV. rcAAV, comprising rep and cap flanked by ITRs, is produced when the rep and cap genes recombine with the ITRs flanking the transgene which results in deletion of the transgene.
A second method that has been used to produce rAAV involves co-transfection of three plasmids into eukaryotic cells. In this method, one plasmid carries the transgene and ITRs (the cis plasmid), a second plasmid encodes the rep and cap genes (the trans plasmid), and the third plasmid encodes the helper virus functions, i.e. adenoviral genes such as E1a, E1b, E2a and E4 (the helper plasmid). The disadvantages of the first method are shared with this method.
A third method involves the use of a packaging cell line such as one including AAV functions rep and cap. See U.S. Pat. Nos. 5,658,785 and 5,837,484 and PCT US98/19463. The packaging cell line may be transfected with a cis plasmid comprising the transgene and ITRs, and infected by wild-type adenovirus (Ad) helper. See U.S. Pat. No. 5,658,785. Alternatively, the packaging cell line may be co-infected by a hybrid Ad/AAV, in which a hybrid Ad vector carries the cis plasmid in the E1 locus (see U.S. Pat. No. 5,856,152), and by a wild-type or mutant Ad that supplies E1. See, e.g., Reference 7. The disadvantage of this method is that it requires making a cell line that expresses sufficient levels of rep and cap, and requires multiple components—including the cell line, the rAAV genome, and an adenovirus—to produce rAAV, which do not lend themselves to easy and convenient downstream manufacturing processes. In addition, some of these packaging cell lines do not produce high levels of rAAV.
A fourth method is provided by a prophetic example in U.S. Pat. No. 5,354,678. The method involves using a recombinant adenovirus in which the rep and cap genes of AAV replace a part of the adenovirus genome not essential for helper virus functions. In this method, an AAV/EBV plasmid vector comprising an rAAV genome is introduced into a cell to produce an rAAV producer cell. It is presumed that the rep gene is driven by its native p5 promoter or by a strong inducible promoter. The recombinant adenovirus comprising rep and cap is then introduced into the cell and their production is induced such that rAAV is produced by the cells. U.S. Pat. No. 5,354,678 does not disclose the levels of rAAV, if any, produced by this method.
As described above, current rAAV production methods are not amenable for production of sufficient rAAV for pharmaceutical applications in a convenient manner. However, the problem of reproducibly generating high levels of substantially homogeneous replication-deficient rAAV by an efficient method that is applicable to large-scale industrial production is solved by the present invention.